Project Summary: Origins of human blood lineages in regenerative medicine Since its original implementation in the 1950s, hematopoietic cell transplantation (HCT) has become the most successful and widely used regenerative cell therapy. Approximately 19,000 patients in the United States received HCT in 2013 and it is currently the standard of care for many myeloid and lymphoid malignancies, as well as blood- and immune-deficiencies. However, there is an urgent need to develop new sources of human hematopoietic material, expand current sources, and manipulate cells in order minimize adverse events and maximize disease free survival. While CD34 expression represents the single most robust cell surface marker to identify hematopoietic stem and progenitor activity in the human less than 1% of even CD34+ cells purified for transplantation represent a long- term engrafting hematopoietic stem cell (HSC). Furthermore, the vast majority of engrafting cells are poorly understood immune effector cells (lymphoid and myeloid-derived) and erythroid precursors (i.e. normoblasts and megakarocytes) that have the most immediate regenerative effect and often persist for a decade or more. A reason for this knowledge gap is human hematopoiesis is based on a discrete hierarchical model of cellular differentiation that starts from a single, multi-potent cell (HSC) and has a conventional framework for identifying cells at each stage. However, these stage-based definitions are functional distinction where less than half of defined cells can actually execute these functions. Moreover, the majority of CD34+ cells in human hematopoietic tissue do not even fit into these discrete populations. Thus, the model is broken! Instead, we are proposing a more realistic view of early human hematopoiesis, where cells differentiate in continuous fashion and lineage fate is based more on probability with most cells following a predictable functional path, while some exhibit higher plasticity for transitioning between lineages. This model offers a more holistic view of hematopoietic immune system development and attempts to reconcile many of the conflicting views and results of the past. These challenges have long existed because elucidating complex cellular relationships in primary human tissue is technically difficult due to limited materials and the ability to manipulate and measure them. Now, the increased parameterization of cytometry has driven the understanding of cellular diversity and molecular processes, respectively. Emerging single-cell proteomic technologies have enabled the simultaneous measurement of an unprecedented large number of cellular features. In particular, CyTOF mass cytometry, a next-generation single cell analysis platform that we pioneered, uses non-biological elemental mass reporters in the place of traditional fluorophores. The throughput of this approach and computational methods it affords facilitates the analysis of complex cellular systems with little need for enrichment or prior knowledge. With these resources at our disposal we will investigate the organization of early human hematopoietic immune cell ontogeny (i.e. formation of effector immune cells and blood cells). In this comprehensive endeavor we will look at this specification of CD34+ cells across primary hematopoietic immune tissues (bone marrow, cord blood, tonsil, thymus, etc.) with the goal of 1) characterizing and developmentally ordering the earliest human hematopoietic cell types, 2) identifying their molecular regulators (transcription factors, epigenetic markers, regulatory enzymes and modifications) inferring their probabilistic relationship with cell fate, and 3) bridging cellular relationships and identities across different hematopoietic cell sources in order to reconcile observed differences and developmental relationships. Thus, by simultaneously examining the single cell proteomic features of multiple primary tissues spanning human hematopoiesis, this application has the lofty goal of providing a predictive snapshot of early human hematopoietic immune developmental process without the need for cellular synchronization or manipulation. More importantly, translation of these approaches can be applied directly to clinical biopsies and blood specimens. Using novel single cell measurement modalities and analytical tools, this process will establish a comprehensive view of human hematopoiesis that will serve as a baseline for understating pathologic perturbations in disease and manipulating these tissues for therapeutic purposes. More broadly, this work will create a roadmap for applying these methods to other tissue types and corrupted developmental processes, having impact on future studies of developing human systems.